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Use of ELISA to test seroprevalence of Toxoplasma gondii in cats
of Tom Green County
Amanda Weaver and Dr. Nicholas Negovetich, Department of Biology
Introduction
Methods
Toxoplasma gondii is a protozoan parasite that infects various mammals and birds.
This parasite is considered zoonotic because it can be transferred from animals to humans
through contact with infected feces or eating undercooked infected meat. When cat feces
containing oocysts are accidentally ingested by other animals, the parasite encysts in the
muscle of the new host, but will not be able to replicate, enter the environment, and infect
other animals. Therefore, cats are essential in the life cycle of T. gondii because they allow
this parasite to spread to other animals in the absence of a direct trophic interaction. For this
reason, I chose to use cats as a model to measure the prevalence of this parasite in Tom
Green County.
I collected samples from four veterinary offices in Tom Green County. These
veterinarians donated aliquots of blood or serum from samples they took throughout
the week for clinical procedures and included the gender and age of the animal from
which the sample came. I picked the samples up at the end of each week, kept track
of which facility they came from (which will allow me to compare the prevalence from
different locations within the county) and kept them in the parasitology lab freezer
until I had 90 samples to run the ELISA test.
There are several ways to test for T. gondii including serological tests (such as
an ELISA), PCR, histological examination, or isolation of the parasite10. I chose to
run an ELISA to test my samples because it is economic, makes use of available
resources (leftover blood samples), and has 96 wells in each plate so I can run many
samples at one time. Since the ELISA tested for IgG antibodies, it detected if the cat
has ever been infected with or exposed to T. gondii, and developed antibodies
against it. Serum samples were considered positive if the absorbance reading at
450 nm equaled or exceeded 2.1 times the absorbance reading of the negative
control. My independent variables of interest include sex (male vs. female), age
(divided into separate age groups), and veterinary clinic.
Collect
Blood
Samples
Results
Run
ELISA
Statistical
Analysis
Compare
to
Literature
Figure 5. Graph showing seroprevalence of 4 different clinics with 95%
bootstrapped confidence intervals. P-value= 0.23
Figure 1. Life cycle of Toxoplasma gondii.
Toxoplasma gondii is a threat to many animals including humans as it causes
fever, confusion, headache, seizures, nausea, and poor coordination in
immunosuppressed individuals. The CDC approximates 22.5% of people in the United
States have been infected, and that number increases to as much as 95% in some parts
of the world2. Although infection with T. gondii usually leads to minimal, if any, symptoms,
it is thought to cause changes in human behavior1. For example, T. gondii is known to
manipulate the behavior of mice to become less fearful of cats which makes them easier
for the cats to consume and helps the parasite move forward in its life cycle. Webster et
al. claim that T. gondii may also affect human behavior by making us more likely to take
risks, i.e., receiving speeding tickets and being involved in work place accidents1. Also,
the presence of antibodies to T. gondii were linked to several cases of schizophrenia,
among other neurological symptoms1. This parasite is most dangerous, though, when a
mother passes it to her child. Congenital toxoplasmosis occurs when women become
infected with T. gondii during their pregnancy, with earlier infection causing more severe
symptoms for the baby including hydrocephalus, mental retardation, and intracerebral
calcifications2. For this reason, it is recommended for a pregnant women to avoid
cleaning litter boxes and to wash their hands thoroughly after picking vegetables from a
garden because cat feces may be present in the soil. Also, meat should be cooked
thoroughly to kill parasites that have encysted in muscle tissue.
Discussion
Before we ran the ELISA, we were expecting 30%-60%3-9 of our samples to be positive
with possibly an even lower number due to the dry conditions of west Texas, which make it
less suitable for oocyst survival6. Also, we were expecting older cats to have a higher
prevalence than younger cats because they have had more time to be exposed to the
parasite5.
However, neither one of our expectations were met which were interesting findings for
us. We believe the higher than expected prevalence could have been caused by several
factors that have had an impact on the parasite’s life cycle including more outdoor than
indoor cats, many intermediate hosts, unforeseen helpful conditions in our environment for
the oocyst to survive such as people watering their yards, and a warmer winter than usual.
The lack of a significance of the prevalence among age groups can be explained by a lack of
variance since 90% of the cats were positive.
Future Directions: If I were able to continue this study in cats, I would want to sample
more cats in our county throughout different times of the year, expand the study to various
parts of Texas, and test for IgM antibodies as well as IgG to show how long the cat has been
infected. Even though I doubt I will be able to continue this study in cats, I may be able to
perform a study on T. gondii in humans while I am in medical school or sometime in the
future.
`
Figure 6. Graph showing seroprevalence of males and Figure 7. Graph showing seroprevalence of five
females with 95% bootstrapped confidence intervals. different age groups with 95% bootstrapped confidence
intervals. P-value= 0.37
P-value= 0.35
Acknowledgements
Figure 2. ELISA kit including wash solution, enzyme
conjugate, substrates, and stop solution.
Figure 3. Using micropipette to
add serum to wells.
Results
We found 90% (81/90) of our samples to be positive for IgG antibodies to T. gondii.
Once we received our data, we ran statistical analysis using R (www.r-project.org) to
determine if sex, age, or clinic had an effect on the seroprevalence of the cats we tested
for this parasite. We didn’t find any significant P-values which is a result of a lack of
variance since we had 90% positive. We also ran an age-sex interaction term to
determine if these two variables combined affected the seroprevalence, but we obtained a
P-value of 0.3 which suggests they do not affect seroprevalence.
We would like to thank the Angelo State University undergraduate faculty mentored and Head
of the River Ranch research grants for helping fund our research. We are also thankful for the
veterinary offices (College Hills, Los Caballos, San Angelo, and River Oaks) that collected
blood samples and data for our project.
Literature Cited
1. Webster, J.P., Kaushik, M., Bristow, G.C., and McConkey, G.A. “Toxoplasma gondii infection, from predation to
schizophrenia: can animal behavior help us understand human behavior?” Journal of Experimental Biology. 216, 99-112.
2013.
2. Center for Disease Control and Prevention. “Parasites- Toxoplasma infection.”
http://www.cdc.gov/parasites/toxoplasmosis/disease.html.
3. Vollaire, M.R., Radecki, S.V., and Lappin, M.R. “Seroprevalence of Toxoplasma gondii antibodies in clinically ill cats in the
United States.” American Journal of Veterinary Research. 66, 874-877.
4. Nutter, F.B., Dubey, J.P., Levine, J.F., Breitshwerdt, E.B., Ford, R.B., and Stoskopf, M.K. “Seroprevalences of antibodies
against Bartonella hensalae and Toxoplasma gondii and fecal shedding of Cryptosporidium spp, Giardia spp, and Toxocara
cati in feral and pet domestic cats.” Journal of American Veterinary Medical Association. 225, 1394-1398.
5. Dubey, J.P., Bhatia, C.R., Lappin, M.R., Ferreira, L.R., Thorn, A., and Kwok, O.C.H. “Seroprevalence of Toxoplasma
gondii and Bartonella spp. Antibodies in cats from Pennsylvania.” Journal of Parasitology. 95, 578-580. 2009.
6. Elmore, S.A., Jones, J.L., Conrad, P.A., Patton, S., Lindsay, D.S., and Dubey, J.P. “Toxoplasma gondii: epidemiology,
feline clinical aspects, and prevention.” Cell Press. 26, 190-196. April 2010.
7. Al-Kappany, Y.M., Rajendran, C., Ferreira, L.R., Kwok, O.C.H, Abu-Elwafa, S.A., Hilali, M., and Dubey, J.P. “High
prevalence of Toxoplasmosis in cats from Egypt: isolation of viable Toxoplasma gondii, tissue distribution, and isolate
designation. Journal of Parasitology. 96, 1115-1118. 2010.
8. Haddadzadeh, H.R., Khazraiinia, P., Aslani, M., Rezaeian, M., Jamshidi, S., Taheri, M., and Bahonar, A. “Seroprevalence
of Toxoplasma gondii in stray and household cats in Tehran.” Veterinary Parasitology. 138, 211-216.
9. Castillo-Morales, V.J., Viana, K.Y.A., Guzman-Marin, E.S., Jimenez-Coello, M., Segura-Correa, J.C., Aguilar-Caballero,
A.J., and Ortego-Pacheco, A. “Prevalence and Risk Factors of Toxoplasma gondii Infection in Domestic Cats from the
Tropics of Mexico Using Serological and Molecular Tests.” Interdisciplinary Perspectives on Infectious Diseases. 2012,
Article ID 529108.
Figure 4. Picture of the 96 well plate after our ELISA was
complete. Yellow is positive and clear is negative.
10. Montoya, J.G. “Laboratory Diagnosis of Toxoplasma gondii Infection and Toxoplasmosis.” Journal of Infectious Diseases.
185, 573-582. 2002.